Molecular markers of plant embryogenesis

ABSTRACT

The present invention relates generally to a molecular marker for a plant physiological process and more particularly for plant embryogenesis. The molecular marker is, in one form, a genetic sequence from a monocot plant such as but not limited to oil-palm plants. In another form, the molecular marker is a polypeptide encoded by said genetic sequence. More particularly, the molecular marker of the present invention enables embryogenic tissue to be detected in vitro. The early detection of embryogenic tissue enables non-embryogenic tissue to be discarded. The ability to detect embryogenesis facilitates maximization of embryogenic potential. The present invention further contemplates a molecular marker comprising in one form a sequence of nucleotides encoding an antioxidant or in another form a sequence of amino acids defining a polypeptide having antioxidant activity. The antioxidant according to this aspect of the present invention is particularly useful in tablet or cream form as an anti-aging agent. The molecular markers of the present invention therefore also have uses in the inhibition or retardation of apoptotic processes. Such an effect has benefits in both plant and animal cells. The present invention further contemplates a promoter sequence encoding the molecular marker and its use in generating male sterile plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/788,725, filed on May 27, 2010, which is a divisionalapplication of Ser. No. 10/028,346, filed on Dec. 20, 2001, whichbenefits Australian Provisional Application No. PR2213/00, filed Dec.20, 2000.

FIELD OF THE INVENTION

The present invention relates generally to a molecular marker for aplant physiological process and more particularly for plantembryogenesis. The molecular marker is, in one form, a genetic sequencefrom a monocot plant such as but not limited to oil-palm plants. Inanother form, the molecular marker is a polypeptide encoded by saidgenetic sequence. More particularly, the molecular marker of the presentinvention enables embryogenic tissue to be detected in vitro. The earlydetection of embryogenic tissue enables non-embryogenic tissue to bediscarded. The ability to detect embryogenesis facilitates maximizationof embryogenic potential. The present invention further contemplates amolecular marker comprising in one form a sequence of nucleotidesencoding an antioxidant or in another form a sequence of amino acidsdefining a polypeptide having antioxidant activity. The antioxidantaccording to this aspect of the present invention is particularly usefulin tablet or cream form as an anti-aging agent. The molecular markers ofthe present invention therefore also have uses in the inhibition orretardation of apoptotic processes. Such an effect has benefits in bothplant and animal cells. The present invention further contemplates apromoter sequence encoding the molecular marker and its use ingenerating male sterile plants.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as 15179.seq.txt of16 KB, created on Jan. 13, 2005, and submitted to the United StatesPatent and Trademark Office via first class mail, is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any othercountry.

The study of plant embryogenesis has been regarded as fundamental tounderstanding plant development. It is during embryogenesis thatmeristems and basic plant tissue systems are established. Basically,embryogenesis involves two main processes: the induction of embryogenicpotential and the expression of the embryogenesis programme. Because ofthe totipotency of the plant cells, each cell has the capability tobecome embryogenic and to develop into a whole plant.

Recent advances in micropropagation and manipulation of tissue cultureconditions has led to the possibility of multiplying vegetatively manyplant species efficiently and rapidly in vitro. For many commercialproduction systems, conventional plant breeding and seed productionmethods are slow and therefore limit the ability to realize the maximumpotential of selected genotypes. However, the development ofeconomically-viable propagation systems necessitates the existence ofefficient methods of embryo- or organogenesis. Such methods have beengenerated for many, but not all species.

There are very high demands for oil-palm. Accordingly, a major area ofstudy in the oil-palm industry seeks to find improved ways to increaseoil yield. With the ability to maintain uniformity of planting materialsin tissue culture, improvements in yield of up to 20% may be able to berealized. In the case of oil-palm, however, little is known about thebiology of somatic embryogenesis despite the economic importance of thecrop and work to date has resulted in average rates of in vitroembryogenesis of only 6% (Wooi, 1995). Such low rates are inconsistentwith an economically viable system.

Most of the earlier studies concentrated on the development ofmethodologies for the initiation and production of somatic embryos(Jones, 1974; Ahee et al., 1981; Pannetier et al., 1981). These groupsworked mainly on the manipulation of phytohormones in the media as wellas on introducing tissues with better clonability to further improve theprocess. Schwendiman and colleagues (1988) carried out histologicalanalysis of somatic embryogenesis from leaf-derived callus, detailingthe emergence of callus and the subsequent formation of somatic embryos,with shoot and root apices. Not long before that, Turnham and Northcote(1982) investigated the occurrence of biochemical indicators that areuseful in the prediction of embryogenic potential.

More recently, the importance of understanding molecular switches, thatoccur in somatic cells and induce them to become embryogenic, has beenhighlighted (Dudits et al., 1995).

In this regard, the rapid introduction of and improvements inrecombinant DNA technologies has greatly facilitated the study of plantdevelopment and provided researchers with sophisticated precision toolsfor investigating underlying molecular mechanisms.

There is a need to develop an effective and efficient method for theproduction of somatic embryos and new approaches to be brought to bearin attempts to realize that end.

In work leading up to the present invention, the inventors sought toidentify underlying factors involved in the induction of embryogenesis.In so doing the inventors located and isolated a polynucleotide sequencewhich was surprisingly found to be expressed only in zygotic embryo andembryogenic callus. The polynucleotide sequence or an amino acid encodedthereby of the present invention is useful inter alia as a means ofdiscriminating embryogenic from non-embryogenic material. The molecularmarker represents a member of a new class of molecules from monocotplants such as but not limited to oil-palm and related plants.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1, <400>2, etc. A sequence listing isprovided after the claims.

The present invention provides a developmentally-regulated nucleic acidmolecule designated herein as OPEm1. The nucleic acid molecule comprisesa nucleotide coding sequence substantially as set forth in SEQ ID NO:1.Additional 3′ and 5′ sequences are provided in SEQ ID NO:3. The nucleicacid molecule is expressed only in zygotic embryo and embroyonic callusto produce a polypeptide comprising the amino acid sequence set forth inSEQ ID NO:2 (corresponding to SEQ ID NO:1) or SEQ ID NO:4 (correspondingto SEQ ID NO:3). The identification of the nucleic acid molecule permitsthe discrimination of plant tissue at different developmental stages.The nucleic acid molecule, therefore, permits identification of a plantphysiological process or tissue or other plant material associated witha plant physiological process.

The nucleic acid molecule and/or the polypeptide encoded thereby of thepresent invention may be used as a means of discriminating embryogenicfrom non-embryogenic material in plants, in particular monocot plantsand even more particularly in oil-palm and related plants. The presentinvention provides a nucleic acid molecule, recombinant and purifiednaturally-occurring polypeptides, antibodies to the polypeptides as wellas transgenic and genetically-modified plants. Furthermore, thepolypeptides of the present invention also have anti-apoptoticproperties, making them useful in the preparation of pharmaceuticalcompositions for use as anti-ageing agents.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule encoding a polypeptide comprising an amino acidsequence substantially as set forth in SEQ ID NO:2 or an amino acidsequence having at least about 71% similarity to SEQ ID NO:2, whereinsaid polypeptide is present in plant zygotic embryos or embryogeniccallus and is substantially not present in non-embryogenic tissue.

Another aspect of the present invention provides an isolated nucleicacid molecule comprising a sequence of nucleotides substantially as setforth in SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or anucleotide sequence having at least about 71% similarity to SEQ ID NO:1or SEQ ID NO:3 or its complementary form or a nucleotide sequencecapable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form under low stringency conditions.

A further aspect of the present invention provides an isolated nucleicacid molecule capable of discriminating embryogenic from non-embryogenicmaterial, wherein said nucleic acid molecule comprises a sequence ofnucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 orits complementary form, or a nucleotide sequence having at least about71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formor a nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form under low stringency conditions.

Yet another aspect of the present invention provides an isolated nucleicacid molecule comprising a polynucleotide sequence substantially as setforth in SEQ ID NO:1 or SEQ ID NO:3.

Still another aspect of the present invention provides a geneticconstruct comprising a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence substantially as set forth in SEQ IDNO:2 or an amino acid sequence having at least about 71% similarity toSEQ ID NO:2, wherein said polypeptide is present in plant zygoticembryos or embryogenic callus and is substantially not present innon-embryogenic tissue.

Even still another spect of the present invention provides a geneticconstruct comprising a nucleic acid molecule comprising a sequence ofnucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 orits complementary form, or a nucleotide sequence having at least about71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formor a nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form under low stringency conditions.

Even yet another aspect of the present invention provides a vectorcomprising a nucleic acid molecule capable of discriminating embryogenicfrom non-embryogenic material, wherein said nucleic acid moleculecomprises a sequence of nucleotides substantially as set forth in SEQ IDNO:1 or SEQ ID NO:3 or its complementary form, or a nucleotide sequencehaving at least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or anucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3or its complementary form under low stringency conditions.

Another aspect of the instant invention provides a host cell comprisinga nucleic acid molecule encoding a polypeptide comprising an amino acidsequence substantially as set forth in SEQ ID NO:2 or an amino acidsequence having at least about 71% similarity to SEQ ID NO:2, whereinsaid polypeptide is present in plant zygotic embryos or embryogeniccallus and is substantially not present in non-embryogenic tissue.

A further aspect of the present invention provides an isolatedpolypeptide or biologically-active fragment thereof or a variant orderivative of these, said polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:2 or an amino acid sequence having at least about71% similarity to SEQ ID NO:2, wherein said polypeptide is present inplant zygotic embryos or embryogenic callus and is substantially notpresent in non-embryogenic tissue.

Yet another aspect of the present invention is directed to an isolatedpolypeptide comprising a sequence of amino acids encoded by thenucleotide sequence substantially as set forth in SEQ ID NO:1 or SEQ IDNO:3 or a nucleotide sequence having at least about 71% similarity toSEQ ID NO:1 or SEQ ID NO:3 or its complementary form or a nucleotidesequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form under low stringency conditions.

Still another aspect of the present invention provides a method forproducing a recombinant polypeptide in a host cell or tissue, saidmethod comprising introducing into the said cell or tissue an expressionvector comprising a nucleic acid molecule wherein said nucleic acidmolecule comprises a sequence of nucleotides substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or a nucleotidesequence having at least about 71% similarity to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions wherein said nucleic acid molecule isoperably linked to one or more regulatory sequences such that thenucleic acid molecule is capable of being expressed in said cell ortissue.

Even still another aspect of the invention provides a method formodulating apoptotic processes in a cell or tissue, said methodcomprising introducing into said cell or tissue an expression vectorcomprising a nucleic acid molecule, said nucleic acid moleculecomprising a sequence of nucleotides substantially as set forth in SEQID NO:1 or SEQ ID NO:3 or its complementary form, or a nucleotidesequence having at least about 71% similarity to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions wherein said nucleic acid molecule isoperably linked to one or more regulatory sequences such that thenucleic acid molecule is capable of being expressed in said cell ortissue.

Even yet another aspect of the invention provides a method formodulating apoptotic processes in a cell, said method comprisingadministering to said cell an apoptotic process-controlling effectiveamount of a recombinant polypeptide, said polypeptide comprising theamino acid sequence set forth in SEQ ID NO:2 or an amino acid sequencehaving at least about 71% similarity to SEQ ID NO:2, said administrationbeing for a time and under conditions sufficient to modulate apoptosis.

Another aspect of the invention provides a method for detectingembryogenic plant material, said method comprising immobilizing a sampleputatively containing RNA from the material to be screened on a solidsupport and contacting said immobilized RNA with a labelled nucleotidesequence capable of hybridizing to all or part of an mRNA transcriptcorresponding to the nucleotide sequence set forth in SEQ ID NO:1 or SEQID NO:3 or their derivatives or homologues as defined herein and thendetecting the presence of said label.

A further aspect of the present invention contemplates an antibody to apolypeptide, said polypeptide comprising a sequence of amino acidssubstantially as set forth in SEQ ID NO:2 or an amino acid sequencehaving at least 71% similarity to SEQ ID NO:2, wherein said polypeptideis present in plant zygotic embryos or embryogenic callus and issubstantially not present in non-embryogenic tissue.

Yet another aspect of the present invention contemplates a method fordetecting a polypeptide which is indicative of the presence ofembryogenic tissue in oil-palm or related plants, said method comprisingcontacting the tissue or an extract thereof with an antibody specificfor said polypeptide or its derivatives or homologues for a time andunder conditions sufficient for an antibody-polypeptide complex to form,and then detecting said complex.

Still another aspect of the present invention contemplates apharmaceutical composition comprising the polypeptide having an aminoacid sequence as set forth in SEQ ID NO:2 or a functional homologuethereof or a molecule having at least 71% similarity to SEQ ID NO:2 andone or more pharmaceutically-acceptable carriers and/or diluents.

Even still another aspect of the present invention is directed to aregenerated differentiated plant comprising a nucleic acid moleculeencoding a polypeptide comprising an amino acid sequence substantiallyas set forth in SEQ ID NO:2 or an amino acid sequence having at leastabout 71% similarity to SEQ ID NO:2, wherein said polypeptide is presentin plant zygotic embryos or embryogenic callus and is substantially notpresent in non-embryogenic tissue.

Even yet another aspect of the present invention is directed to aregenerated differentiated plant comprising a nucleic acid molecule,wherein said nucleic acid molecule comprises a sequence of nucleotidessubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form, or a nucleotide sequence having at least about 71%similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form or anucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3or its complementary form under low stringency conditions.

Another aspect of the present invention provides an isolated nucleicacid molecule having promoter activity wherein, in its naturallyoccurring form, the promoter is operably linked to a nucleotide sequencesubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or a nucleotidesequence complementary thereto or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions.

SUMMARY OF SEQUENCE IDENTIFIERS SEQUENCE IDENTIFER DESCRIPTION SEQ IDNO: 1 Nucleotide coding sequence of embryogenic specific polypeptidefrom oil-palm (OPEm1) SEQ ID NO: 2 Amino acid sequence of embryogenicspecific polypeptide from oil-palm (OPEm1); corresponds to SEQ ID NO: 1SEQ ID NO: 3 Nucleotide coding sequence of embryogenic specificpolypeptide from oil-palm (OPEm1) with 5′ and 3′ non-transcribedsequences SEQ ID NO: 4 Amino acid sequence of embryogenic specificpolypeptide from oil-palm (OPEm1); corresponds to SEQ ID NO: 3 SEQ IDNO: 5 Oligonucleotide primer AGL15AtF SEQ ID NO: 6 Oligonucleotideprimer AGL15AtR SEQ ID NO: 7 Amino acid sequence of 1-Cys peroxiredoxinfrom Hordeum vulgare (barley) [HvPer1] SEQ ID NO: 8 Amino acid sequenceof 1-Cys peroxiredoxin from Arabidopsis thaliana (thalecress) [AtPer1]SEQ ID NO: 9 Amino acid sequence of 1-Cys peroxiredoxin from Brassicacampestris (Chinese cabbage) [C2CPRX]

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a is a photographic representation showing reverse transcriptionof zygotic embryo total RNA with primers AGL15AtF and AGL15AtR resultingin the production of two bands, designated RTPCR1 (˜562 bp) and RTPCR2(˜501 bp) (left). The bands were excised and purified (right).

FIG. 1 b is a photographic representation showing Northern analysis ofRTPCR1 and RTPCR2. RTPCR1 was exclusively expressed in embryogenicmaterials (ZE and EC). However, RTPCR2 was constitutively expressed. ZE:zygotic embryo; EC: embryogenic calli; NEC: non-embryogenic calli; YL:young leaves.

FIG. 2 a is a photographic representation showing Northern analysis ofOPEm1 (top) and expression of 18S ribosomal cDNA (bottom) as control.Each lane contains 10 μg of total RNA from different types of tissues.Lane 1: embryogenic calli from clone FC1454; Lane 2: embryogenic calliof clone FC1454 that have lost their embryogenic potential (ENP); Lane3: embryogenic calli from FC1501; Lane 4: ENP of FC1501; Lane 5:Non-embryogenic calli of FC1501. Lane 6: embryogenic calli of FC1509;Lane 7: ENP of FC1509; Lane 8: non-embryogenic calli of FC1509; Lane 9:embryogenic calli from early suspension cultures; Lane 10: suspensioncultures; Lane 11: white embryoids; Lane 12: green embryoids; Lane 13:bipolar structures; Lane 14: immature 12 WAA zygotic embryos; Lane 15:mature 15 WAA zygotic embryos; Lane 16: vegetative meristem; Lane 17:inflorescence from frond 17 and Lane 18: young leaves.

FIG. 2 b is a graphical representation showing hydropathy plot of OPEm1with a calculated pI of 7.48.

FIG. 2 c is a photographic representation showing Southern analysis ofOPEm1. Each lane contains 10 μg genomic DNA digested with: Lane 1:EcoRI; Lane 2: BamHI; Lane 3: HindIII; Lane 4: KpnI; Lane 5: NotI; Lane6: SfiI; Lane 7: SpeI and Lane 8: StuI. The digests were run alongside a1 kb DNA molecular weight marker (Promega).

FIG. 3 is the nucleotide and deduced amino acid sequences of OPEm1.

FIG. 4 a is photographic representation showing a 3-D structure of themonomer unit of the human peroxiredoxin, C91S-hORF6. Each monomerconsists of 224 amino acids with two domains. Domain I is the largerN-terminal domain and Domain II is the smaller C-terminal domain.

FIG. 4 b is a photographic representation showing that humanperoxiredoxin exists as a tightly associated dimer.

FIG. 4 c is a photographic representation showing the deduced 3-Dstructure of OPEm1, which has very high similarity to the humanperoxiredoxin.

FIG. 4 d is a schematic representation depicting a topology diagram ofC91S-hORF6 monomer. The shaded area corresponds to the thioredoxin fold.

FIG. 5 is the sequence alignment of OPEm1 with examples of 1-Cys and2-Cys peroxiredoxins. The ‘*’ denotes a single fully conserved residue.The ‘:’ denotes conservation of strong groups. The ‘.’ conservation ofweak groups. Those without any symbol denote no consensus (CLUSTALW,Biology Workbench Version 3.2, University of Illinois, 1999). The aminoacid sequences were obtained from Genbank: HvPer1 (Hordeum vulgare,barley, P52572), AtPer1 (Arabidopsis thaliana, thalecress, CAA63909) andC2CPRX (Brassica campestris L. ssp. pekinensis, chinese cabbage).

FIG. 6 is the sequence alignment of OPEm1 with examples of other membersof 1-Cys peroxiredoxin. The ‘*’ denotes a single fully conservedresidue. The ‘:’ denotes conservation of strong groups. The ‘.’ denotesconservation of weak groups. Those without any symbols denote noconsensus. (CLUSTALW, Biology Workbench Version 3.2, University ofIllinois, 1999). The peroxiredoxin amino acid sequences were obtainedfrom Genbank: HvPer1 (Hordeum vulgare, barley, P52572) and AtPer1(Arabidopsis thaliana, thalecress, CAA63909). The ‘#’ and ‘@’ denote thepositively charged residue His 38 and Arg 128 respectively, which areall found close to the Cys 46. The PVCT region represents a specificcharacteristic of the 1-Cys peroxiredoxin. The basic residues at theterminal end of the 1-Cys peroxiredoxin align to the nuclearlocalization signal (NLS) region that is not present in OPEm1. Acoloured version of this Figure where the PVCT region is in blue and thebasic region is in red is available from the Applicant upon request.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification of adevelopmentally-regulated nucleic acid molecule. The identification ofthe nucleic acid molecule permits the discrimination of plant tissue atdifferent developmental stages. The nucleic acid molecule, therefore,permits identification of a plant physiological process or tissue orother plant material associated with a plant physiological process. Moreparticularly, the polynucleotide sequence is expressed only in zygoticembryo and embryogenic callus. The nucleic acid molecule and/or an aminoacid sequence encoded thereby of the present invention may be used as ameans of discriminating embryogenic from non-embryogenic material inplants, in particular monocot plants and even more particularly inoil-palm and related plants. The term “material” includes cells, tissue,clusters of cells, callus, organelles, seeds, pollen and other plantparts. The nucleic acid molecule of the present invention and an aminoacid sequence encoded thereby are both referred to herein as a“molecular marker”. Reference herein to a “molecular marker” is not toimpart any limitation as to its structure, location in a cell or itsuse.

Reference to the term “discriminating” in relation to embryogenic andnon-embryogenic tissue includes reference to the determination of astrong likelihood that certain tissue is embryogenic as distinct fromnon-embryogenic on the basis of the presence of the subject nucleic acidmolecule or its expression product. Reference to a “determination”includes reference to a “prediction” or other reasoned deduction.Embryogenic material includes inter alia zygotic embryo and embryogeniccallus material.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule encoding a polypeptide comprising an amino acidsequence substantially as set forth in SEQ ID NO:2 or an amino acidsequence having at least about 71% similarity to SEQ ID NO:2, whereinsaid polypeptide is present in plant zygotic embryos or embryogeniccallus and is substantially not present in non-embryogenic tissue.

In a related embodiment, the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides substantiallyas set forth in SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, ora nucleotide sequence having at least about 71% similarity to SEQ IDNO:1 or SEQ ID NO:3 or its complementary form or a nucleotide sequencecapable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form under low stringency conditions.

Preferably the nucleic acid molecule is regulated developmentally suchthat its presence may be used as a means of discriminating embryogenicfrom non-embryogenic material.

Accordingly, in a preferred embodiment, the present invention providesan isolated nucleic acid molecule capable of discriminating embryogenicfrom non-embryogenic material, wherein said nucleic acid moleculecomprises a sequence of nucleotides substantially as set forth in SEQ IDNO:1 or SEQ ID NO:3 or its complementary form, or a nucleotide sequencehaving at least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 orits complementary form or a nucleotide sequence capable of hybridizingto SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under lowstringency conditions.

The term “nucleic acid molecule” includes a polynucleotide, nucleotideor genetic sequence such as, but not limited to, mRNA, RNA, cRNA, cDNAor DNA. Reference to a DNA molecule includes genomic DNA.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated nucleic acid molecule” as used herein refers to apolynucleotide sequence, which has been purified from the sequenceswhich flank it in a naturally-occurring state, e.g., a DNA fragmentwhich has been removed from the sequences which are normally adjacent tothe fragment.

The isolated nucleotide sequence of the present invention also extendsto derivatives including, mutants and homologues of the said sequence.

By “derivative” is meant any single or multiple nucleotide deletions,additions or substitutions as well as mutants, fragments, portions orparts of said isolated nucleic acid molecule. All such deletions,additions, substitutions, mutants, fragments, portions, or parts areencompassed by the term “derivative”. Particularly useful derivativesinclude alterations to the 5′ end portion of the polynucleotide sequenceor the 3′ end portion or a nucleotide sequence spanning the 5′ and 3′portions. Synthetic derivatives may also be useful, for example, indiagnostic assays. A derivative also conveniently includes apolynucleotide sequence having less than 100% identity with thenucleotide sequence set forth in SEQ ID NO:1, but which is capable ofhybridizing thereto or its complementary form under low stringencyconditions.

Terms such as “hybridization”, “hybridizing” and the like are usedherein to denote the pairing of complementary nucleotide sequences toproduce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary basesequences are those sequences that are related by the base-pairingrules. In DNA, A pairs with T and C pairs with G. In RNA, U pairs with Aand C pairs with G. In this regard, the terms “match” and “mismatch” asused herein refer to the hybridization potential of paired nucleotidesin complementary nucleic acid strands. Matched nucleotides hybridizeefficiently, such as the classical A-T and G-C base pair mentionedabove. Mismatches are other combinations of nucleotides that do nothybridize efficiently.

“Stringency” as used herein, refers to the temperature and ionicstrength conditions, and presence or absence of certain organicsolvents, during hybridization and washing procedures. The higher thestringency, the higher will be the degree of complementarity betweenimmobilized target nucleotide sequences and the labelled probepolynucleotide sequences that remain hybridized to the target afterwashing.

“Stringency conditions” refers to temperature and ionic conditions underwhich only nucleotide sequences having a high frequency of complementarybases will hybridize. The stringency required is nucleotide sequencedependent and depends upon the various components present duringhybridization and subsequent washes, and the time allowed for theseprocesses. Generally, in order to maximize the hybridization rate,non-stringent hybridization conditions are selected: about 20 to 25° C.lower than the thermal melting point (T_(m)). The T_(m) is thetemperature at which 50% of specific target sequence hybridizes to aperfectly complementary probe in solution at a defined ionic strengthand pH. Generally, in order to require at least about 85% nucleotidecomplementarity of hybridized sequences, highly stringent washingconditions are selected to be about 5 to 15° C. lower than the T_(m). Inorder to require at least about 71% nucleotide complementarity ofhybridized sequences, moderately stringent washing conditions areselected to be about 15 to 30° C. lower than the T_(m). Highlypermissive (low stringency) washing conditions may be as low as 50° C.below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences.

Reference herein to a low stringency includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is at from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about0.15 M salt for washing conditions. In general, washing is carried outT_(m)=69.3+0.41 (G+C) % (Marmur and Doty, 1962). However, the T_(m) of aduplex DNA decreases by 1° C. with every increase of 1% in the number ofmismatch base pairs (Bonner and Laskey, 1974). Formamide is optional inthese hybridization conditions. Accordingly, particularly preferredlevels of stringency are defined as follows: low stringency is 6×SSCbuffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSCbuffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.;high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of atleast 65° C.

Suitably, the isolated nucleic acid molecule has at least greater than70% (for example, 71%), preferably at least about 75%, more preferablyat least about 80%, more preferably yet at least about 85%, still morepreferably at least about 90% and even still more preferably at leastabout 95% or above (e.g. 96% or 97% or 98% or 99%) sequence similarityto the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. In a particularly preferredembodiment, nucleotide and sequence comparisons are made at the level ofidentity rather than similarity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence similarity”, “sequence identity”,“percentage of sequence similarity”, “percentage of sequence identity”,“substantially similar” and “substantial identity”. A “referencesequence” is at least 12 but frequently 15 to 18 and often at least 25or above, such as 30 monomer units, inclusive of nucleotides and aminoacid residues, in length. Because two polynucleotides may each comprise(1) a sequence (i.e. only a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment oftypically 12 contiguous residues that is compared to a referencesequence. The comparison window may comprise additions or deletions(i.e. gaps) of about 20% or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by computerized implementations ofalgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science DriveMadison, Wis., USA) or by inspection and the best alignment (i.e.resulting in the highest percentage homology over the comparison window)generated by any of the various methods selected. Reference also may bemade to the BLAST family of programs as for example disclosed byAltschul et al. (1997). A detailed discussion of sequence analysis canbe found in Unit 19.3 of Ausubel et al. (1998).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala,Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp,Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For the purposes of the present invention, “sequenceidentity” will be understood to mean the “match percentage” calculatedby the DNASIS computer program (Version 2.5 for windows; available fromHitachi Software engineering Co., Ltd., South San Francisco, Calif.,USA) using standard defaults as used in the reference manualaccompanying the software. Similar comments apply in relation tosequence similarity.

A particularly preferred embodiment of the instant invention provides anisolated nucleic acid molecule comprising a polynucleotide sequencesubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3. Although thepresent invention is particularly exemplified with respect to oil-palm,this is done with the understanding that the instant inventionencompasses any monocot plant. Reference herein to a monocot includesany member of the plant family Gramineae, Palmae, Juncaceae and Achenes,but is not limited to cereals, grasses, maize, sugar cane, oats, wheat,barley as well as oil-palm.

In a convenient embodiment, reference to a nucleic acid moleculeincludes reference to a “gene”. The term “gene” is used in its broadestsense and includes reference to a polynucleotide sequence such as a cDNAcorresponding to the exons of a gene. Accordingly, reference herein to aAgene≅ is to be taken to include:—

-   (i) a classical genomic gene consisting of transcriptional and/or    translational regulatory sequences and/or a coding region and/or    non-translated sequences (i.e. introns, 5′- and 3′-untranslated    sequences); or-   (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons)    and 5′- and 3′-untranslated sequences of the gene; and/or-   (iii) a structural region corresponding to the coding regions (i.e.    exons) optionally further comprising untranslated sequences and/or a    heterologous promoter sequence which consists of transcriptional    and/or translational regulatory regions capable of conferring    expression characteristics on said structural region.

The term “gene” is also used to describe synthetic or fusion moleculesencoding all or part of a functional product, in particular, a sense orantisense mRNA product or a peptide, oligopeptide or polypeptide or abiologically-active protein. Reference to a “gene” also includesreference to a “synthetic gene”.

The term “synthetic gene” refers to a non-naturally occurring gene ashereinbefore defined which preferably comprises at least one or moretranscriptional and/or translational regulatory sequences operablylinked to a structural gene sequence.

The term “structural gene” shall be taken to refer to a nucleotidesequence, which is capable of being transmitted to produce mRNA andoptionally, encodes a peptide, oligopeptide, polypeptide or biologicallyactive protein molecule. Those skilled in the art will be aware that notall mRNA is capable of being translated into a peptide, oligopeptide,polypeptide or protein; for example, if the mRNA lacks a functionaltranslation start signal or alternatively, if the mRNA is antisensemRNA. The present invention clearly encompasses synthetic genescomprising nucleotide sequences, which are not capable of encodingpeptides, oligopeptides, polypeptides or biologically-active proteins.In particular, the present inventors have found that such syntheticgenes may be useful, for example, in diagnostic assays of geneexpression in cells, tissues or organs of a eukaroytic organism.

The term “structural gene region” refers to that part of a syntheticgene, which is expressed in a cell, tissue or organ under the control ofa promoter sequence to which it is operably connected. A structural generegion may be operably under the control of a single promoter sequenceor multiple promoter sequences. Accordingly, the structural gene regionof a synthetic gene may comprise a nucleotide sequence, which is capableof encoding an amino acid sequence or is complementary thereto. In thisregard, a structural gene region, which is used in the performance ofthe instant invention, may also comprise a nucleotide sequence whichencodes an amino acid sequence yet lacks a functional translationinitiation codon and/or a functional translation stop codon and, as aconsequence, does not comprise a complete open reading frame. In thepresent context, the term “structural gene region” also extends to anon-coding nucleotide sequences, such as 5′-upstream or 3′-downstreamsequences of a gene which would not normally be translated in aeukaryotic cell which expresses said gene.

In another aspect, the invention is directed to a vector comprising thenucleic acid molecule as broadly described above. The vector comprisingthe nucleic acid molecule may be in isolated form or may exist as anextrachromosomal element or all or part of the vector may be integratedinto the genome of a host cell. The vector may also be packaged for salein a kit with instructions for use inter alia as a diagnostic agent orin an assay system.

In a preferred embodiment, the instant invention provides a vectorcomprising a nucleic acid molecule having a sequence of nucleotidessubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form, or a nucleotide sequence having at least about 71%similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form or anucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3or its complementary form under low stringency conditions.

A particularly preferred embodiment of the present invention provides avector comprising a nucleic acid molecule capable of discriminatingembryogenic from non-embryogenic material, wherein said nucleic acidmolecule comprises a sequence of nucleotides substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or a nucleotidesequence having at least about 71% similarity to SEQ ID NO:1 or SEQ IDNO:3 or a nucleotide sequence capable of hybridizing to SEQ ID NO:1 orSEQ ID NO:3 or its complementary form under low stringency conditions.

Furthermore, the vector may comprise a nucleic acid molecule which whentranscribed generates a mRNA which is antisense relative to thetranscript generated by SEQ ID NO:1 or SEQ ID NO:3 or its relatedsequence. A related sequence includes a nucleotide sequence having atleast about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form or a nucleotide sequence capable of hybridizing toSEQ ID NO:1 or SEQ ID NO:3 or its complementary form under lowstringency conditions. A related sequence, therefore, includes aderivative and homologue.

In a further related embodiment, the invention is directed to a vectorcomprising a polynucleotide sequence as broadly described above whereinthe polynucleotide sequence is operably linked to one or more regulatorysequences, including but not limited to a promoter sequence and/or atranscription terminator sequence.

By “operably linked” is meant that transcriptional and translationalregulatory nucleic acids are positioned relative to a functional codingregion in such a manner that the functional coding region is transcribedand optionally the polypeptide is translated. The term “functional”includes a nucleotide sequence which encodes a peptide, polypeptide orprotein, or which exhibits some other function such as but not limitedto binding to DNA or RNA.

By “vector” is meant a nucleic acid molecule, preferably a DNA moleculederived, for example, from a plasmid, bacteriophage, or plant virus,into which a nucleic acid sequence may be inserted or cloned. A vectormay also be a form of genetic construct. A vector preferably containsone or more unique restriction sites and may be capable of autonomousreplication in a defined host cell including a target cell or tissue ora progenitor cell or tissue thereof, or be integrable with the genome ofthe defined host such that the cloned sequence is reproducible.Accordingly, the vector may be an autonomously replicating vector, i.e.a vector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a linear or closedcircular plasmid, an extrachromosomal element, a minichromosome, or anartificial chromosome. The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into a cell, is integrated into the genome of the recipientcell and replicated together with the chromosome(s) into which it hasbeen integrated. A vector system may comprise a single vector orplasmid, two or more vectors or plasmids, which together contain thetotal DNA to be introduced into the genome of the host cell, or atransposon. The choice of the vector will typically depend on thecompatibility of the vector with the cell into which the vector is to beintroduced. The vector may also include a selectable marker such as anantibiotic resistance gene that can be used for selection of suitabletransformants. Examples of such resistance genes are well known to thoseof skill in the art.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences of a classicalgenomic gene, including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner. Apromoter is usually, but not necessarily, positioned upstream or 5′, ora structural gene region, the expression of which it regulates.Furthermore, the regulatory elements comprising a promoter are usuallypositioned within 2 kb of the start site of transcription of the gene.

In the present context, the term “promoter” is also used to describe asynthetic or fusion molecule, or derivative which confers, activates orenhances expression of a nucleic acid molecule in a cell. The term“expression” encompasses transcription to a mRNA molecule alone or bothtranscription and translation to a corresponding amino acid sequence. By“mRNA” is meant either a sense or antisense mRNA molecule.

Preferred promoters may contain additional copies of one or morespecific regulatory elements, to further enhance expression of the sensemolecule and/or to alter the spatial expression and/or temporalexpression of said sense molecule. For example, regulatory elementswhich confer copper inducibility may be placed adjacent to aheterologous promoter sequence driving expression of a sense molecule,thereby conferring copper inducibility on the expression of saidmolecules.

Placing a nucleic acid molecule under the regulatory control of apromoter sequence means positioning the said molecule such thatexpression is controlled by the promoter sequence. Promoters aregenerally positioned 5′ (upstream) to the genes that they control. Inthe construction of heterologous promoter/structural gene combinations,it is generally preferred to position the promoter at a distance fromthe gene transcription start site that is approximately the same as thedistance between that promoter and the gene it controls in its naturalsetting, i.e. the gene from which the promoter is derived. As is knownin the art, some variation in this distance can be accommodated withoutloss of promoter function. Similarly, the preferred positioning of aregulatory sequence element with respect to a heterologous gene to beplaced under its control is defined by the positioning of the element inits natural setting, i.e. the genes from which it is derived. Again, asis known in the art, some variation in this distance can also occur.

Examples of promoters suitable for use in the synthetic genes of thepresent invention include viral, fungal, bacterial, animal and plantderived promoters capable of functioning in plant, animal, insect,fungal, yeast or bacterial cells. The promoter may regulate theexpression of the structural gene component constitutively, ordifferentially with respect to cell, the tissue or organ in whichexpression occurs or, with respect to the developmental stage at whichexpression occurs, or in response to external stimuli such asphysiological stresses, or pathogens, or metal ions, amongst others.

Preferably, the promoter is capable of regulating expression of anucleic acid molecule in a eukaryotic cell, tissue or organ, at leastduring the period of time over which the target gene is expressedtherein and more preferably also immediately preceding the commencementof detectable expression of the target gene in said cell, tissue ororgan.

Accordingly, strong constitutive promoters are particularly useful forthe purposes of the present invention or promoters, which may be inducedby virus infection or the commencement of target gene expression.

Plant-operable and animal-operable promoters are particularly preferredfor use in the construct of the present invention. Examples of preferredpromoters include the bacteriophage T7 promoter, bacteriophage T3promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 latepromoter, SV40 early promoter, RSV-LTR promoter, CMV IF promoter, CaMV35S promoter, SCSV promoter, SCBV promoter and the like.

In consideration of the preferred requirement for high-level expressionwhich coincides with expression of the target gene or precedesexpression of the target gene, it is highly desirable that the promotersequence is a constitutive strong promoter such as the CMV-IE promoteror the SV40 early promoter sequence, the SV40 late promoter sequence,the CaMV 35S promoter, or the SCBV promoter, amongst others. Thoseskilled in the art will readily be aware of additional promotersequences other than those specifically described.

In the present context, the terms “in operable connection with” or“operably under the control” or similar shall be taken to indicate thatexpression of the structural gene region or multiple structural generegion is under the control of the promoter sequence with which it isspatially connected; in a cell, tissue, organ or whole organism.

The construct preferably contains additional regulatory elements forefficient transcription, for example, a transcription terminationsequence.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active inplant cells are known and described in the literature. They may beisolated from bacteria, fungi, viruses, animals and/or plants orsynthesized de novo.

As with promoter sequences, the terminator may be any terminatorsequence which is operable in the cells, tissues or organs in which itis intended to be used.

The present invention further extends to the promoter of the genesequence defined by SEQ ID NO:1 or SEQ ID NO:3. Accordingly, anotheraspect of the present invention contemplates an isolated nucleic acidmolecule having promoter activity wherein, in its naturally occurringform, the promoter is operably linked to a nucleotide sequencesubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or a nucleotidesequence complementary thereto or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions.

Examples of terminators particularly suitable for use in the syntheticgenes of the present invention include the SV40 polyadenylation signal,the HSV TK polyadenylation signal, the CYC1 terminator, ADH terminator,SPA terminator, nopaline synthase (NOS) gene terminator of Agrobacteriumtumefaciens, the terminator of the cauliflower mosaic virus (CaMV) 35Sgene, the zein gene terminator from Zea mays, the Rubisco small subunitgene (SSU) gene terminator sequences, subclover stunt virus (SCSV) genesequence terminators, any rho-independent E. coli terminator, or thelacZ alpha terminator, amongst others.

In a particularly preferred embodiment, the terminator is the SV40polyadenylation signal or the HSV TK polyadenylation signal which areoperable in animal cells, tissues and organs, octopine synthase (OCS) ornopaline synthase (NOS) terminator active in plant cells, tissue ororgans, or the lacZ alpha terminator which is active in prokaryoticcells.

Those skilled in the art will be aware of additional terminatorsequences, which may be suitable for use in performing the invention.Such sequences may readily be used without any undue experimentation.

Another aspect provides a host cell containing the nucleic acid moleculeof the present invention. In one embodiment the said nucleic acidmolecule is conveniently comprised within a vector as hereinbeforedescribed. In another embodiment, all or part of the nucleic acidmolecule of the present invention may be integrated into the DNA of thehost cell. Suitably, the host cell is a bacterium or other prokaryote,or a plant cell or other eukaryote. In a particularly preferredembodiment, the plant is oil-palm or a related plant. A related plant isone which includes a plant having similarity at the genetic,biochemical, immunological, physiological or behavoural levels tooil-palm plants. Genetic similarity, for example, includes similar codonusage, genetic organization and nucleotide similarity (e.g. at leastabout 71% similarity over defined regions).

Accordingly, a further aspect of the instant invention provides a hostcell comprising a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence substantially as set forth in SEQ IDNO:2 or an amino acid sequence having at least about 71% similarity toSEQ ID NO:2, wherein said polypeptide is present in plant zygoticembryos or embryogenic callus and is substantially not present innon-embryogenic tissue.

The present invention also contemplates the production of recombinantproteins, polypeptides or peptides in a host cell. A reference herein to“proteins”, “polypeptides” or “peptides” is a reference to a polymer ofamino acid residues and to variants of the same. The terms “proteins”,“polypeptides” and “peptides” are used interchangeably. The productionof recombinant polypeptides is useful, for example, to generatemolecules for production of antibodies for use as a diagnostic agent oras a potential therapeutic.

Accordingly, another aspect of the present invention provides anisolated polypeptide or biologically-active fragment thereof or avariant or derivative of these, said polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:2 or an amino acid sequence havingat least about 71% similarity to SEQ ID NO:2, wherein said polypeptideis present in plant zygotic embryos or embryogenic callus and issubstantially not present in non-embryogenic tissue.

In a related embodiment, the present invention is directed to anisolated polypeptide comprising a sequence of amino acids encoded by thenucleotide sequence substantially as set forth in SEQ ID NO:1 or SEQ IDNO:3 or a nucleotide sequence having at least about 71% similarity toSEQ ID NO:1 or SEQ ID NO:3 or its complementary form or a nucleotidesequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form under low stringency conditions.

In one embodiment, without wishing to limit the present invention to anyone theory or mode of operation, the polypeptide may be useful as anantioxidant for the modulation of cellular apoptotic processes. Cellwall metabolism involves deposition of insoluble proteins that can beobserved as thickening of the cell wall surrounding the proembryos. Theinsolubization process has been linked to the presence of hydrogenperoxide which accumulates during metabolic processes. Hence, in aparticular embodiment, the nucleic acid molecule of the presentinvention encodes an antiboxidant only found in embryogenic tissues, andwhich may be responsible for protecting proembryos from being destroyedby the accumulation of hydrogen peroxide within cells. In a particularlypreferred embodiment, the polypeptide of the present invention encodes aperoxiredoxin useful for the modulation of cellular apoptotic processes.In another embodiment, the polypeptide is useful as an immunologicalagent to generate antibodies useful as diagnostic markers.

By “biologically active fragment” is meant a fragment of a full-lengthparent polypeptide which fragment retains the activity of the parentpolypeptide. A biologically active fragment may therefore compriseperoxiredoxin activity, which protects tissues from reactive oxygenspecies (ROS). Alternatively, or in addition, the fragment may retainone or more epitopes for generating antibodies therefor. As used herein,the term “biologically active fragment” includes deletion mutants andsmall peptides, for example, of at least 10, preferably at least 20 andmore preferably at least 30 contiguous amino acids, which comprise theabove activities. Peptides of this type may be obtained through theapplication of standard recombinant nucleic acid techniques orsynthesized using conventional liquid or solid phase synthesistechniques. For example, reference may be made to solution synthesis orsolid phase synthesis as described, for example, in Chapter 9 entitled“Peptide Synthesis” by Atherton and Shephard which is included in apublication entitled “Synthetic Vaccines” edited by Nicholson andpublished by Blackwell Scientific Publications. Alternatively, peptidescan be produced by digestion of a polypeptide of the invention withproteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcusV8-protease. The digested fragments can be purified by, for example,high performance liquid chromatographic (HPLC) techniques.

Hence, another aspect of the present invention provides a method forproducing a recombinant polypeptide in a host cell or tissue, saidmethod comprising introducing into the said cell or tissue an expressionvector comprising a nucleic acid molecule wherein said nucleic acidmolecule comprises a sequence of nucleotides substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or a nucleotidesequence having at least about 71% similarity to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions wherein said nucleic acid molecule isoperably linked to one or more regulatory sequences such that thenucleic acid molecule is capable of being expressed in said cell ortissue.

According to another aspect of the invention, there is provided a methodfor modulating apoptotic processes in a cell or tissue, said methodcomprising introducing into said cell or tissue an expression vectorcomprising a nucleic acid molecule, said nucleic acid moleculecomprising a sequence of nucleotides substantially as set forth in SEQID NO:1 or SEQ ID NO:3 or its complementary form, or a nucleotidesequence having at least about 71% similarity to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions wherein said nucleic acid molecule isoperably linked to one or more regulatory sequences such that thenucleic acid molecule is capable of being expressed in said cell ortissue.

In an alternative embodiment, the present invention contemplates amethod for modulating apoptotic processes in a cell, said methodcomprising administering to said cell an apoptotic process-controllingeffective amount of a recombinant polypeptide, said polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:2 or an aminoacid sequence having at least about 71% similarity to SEQ ID NO:2, saidadministration being for a time and under conditions sufficient tomodulate apoptosis.

The terms “modulating” and “modulate” include up-regulating anddown-regulating expression of the subject nucleic acid molecule orlevels of the instant polypeptide. Inducing apoptosis may be useful inthe treatment of plants and animals (including humans) of cancers, gallsand other outgrowths. Preventing apoptosis may be important for treatingneurodegenerative disorders or other necrotic conditions. Accordingly,the present invention further contemplates a composition, such as apharmaceutical composition, comprising the polypeptide of the instantinvention or comprising genetic molecules capable of encoding saidpolypeptide. Such composition generally also comprises one or morepharmaceutically acceptable carriers and/or diluents.

Means of introducing vectors into cells or tissues (i.e. transfecting ortransforming target cells) are well-known to those skilled in the art.

The constructs described supra are capable of being modified further,for example, by the inclusion of marker nucleotide sequences encoding adetectable marker enzyme or a functional analogue or derivative thereof,to facilitate detection of the synthetic gene in a cell, tissue or organin which it is expressed. According to this embodiment, the markernucleotide sequences will be present in a translatable format andexpressed, for example, as a fusion polypeptide with the translationproduct(s) of any one or more of the structural genes or alternativelyas a non-fusion polypeptide.

Those skilled in the art will be aware of how to produce the syntheticgenes described herein and of the requirements for obtaining theexpression thereof, when so desired, in a specific cell or cell-typeunder the conditions desired. In particular, it will be known to thoseskilled in the art that the genetic manipulations required to performthe present invention may require the propagation of a genetic constructdescribed herein or a derivative thereof in a prokaryotic cell such asan E. coli cell or a plant cell or an animal cell.

The constructs of the present invention may be introduced to a suitablecell, tissue or organ without modification as linear DNA, optionallycontained within a suitable carrier, such as a cell, virus particle orliposome, amongst others. To produce a genetic construct, the syntheticgene of the invention is inserted into a suitable vector or opisomemolecule, such as a bacteriophage vector, viral vector or a plasmid,cosmid or artificial chromosome vector which is capable of beingmaintained and/or replicated and/or expressed in the host cell, tissueor organ into which it is subsequently introduced.

Accordingly, another aspect of the present invention provides a geneticconstruct comprising a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence substantially as set forth in SEQ IDNO:2 or an amino acid sequence having at least about 71% similarity toSEQ ID NO:2, wherein said polypeptide is present in plant zygoticembryos or embryogenic callus and is substantially not present innon-embryogenic tissue.

In a related aspect of the invention, there is provided a geneticconstruct which at least comprises a nucleic acid molecule comprising asequence of nucleotides substantially as set forth in SEQ ID NO:1 or SEQID NO:3 or its complementary form, or a nucleotide sequence having atleast about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form or a nucleotide sequence capable of hybridizing toSEQ ID NO:1 or SEQ ID NO:3 or its complementary form under lowstringency conditions and one or more origins of replication and/orselectable marker gene sequences.

Genetic constructs are particularly suitable for the transformation of aeukaryotic cell to introduce novel genetic traits thereto, in additionto the provision of resistance characteristics to viral pathogens. Suchadditional novel traits may be introduced in a separate geneticconstruct or, alternatively, on the same genetic construct whichcomprises the synthetic genes herein described. Those skilled in the artwill recognize the significant advantages, in particular in terms ofreduced genetic manipulations and tissue culture requirements andincreased cost-effectiveness of including genetic sequences which encodesuch additional traits and the synthetic genes described herein in asingle genetic construct.

Usually, an origin of replication or a selectable marker gene suitablefor use in bacteria is physically-separated from those genetic sequencescontained in the genetic construct which are intended to be expressed ortransferred to a eukaryotic cell, or integrated into the genome of aeukaryotic cell.

As used herein, the term “selectable marker gene” includes any genewhich confers a phenotype on a cell on which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a genetic construct of the invention ora derivative thereof.

Suitable selectable marker genes contemplated herein include theampicillin-resistance gene (Amp^(r)), tetracycline-resistance gene(Tc^(r)), bacterial kanamycin-resistance gene (Kan^(r)), is the zeocinresistance gene (Zeocin is a drug of the bleomycin family which is trademark of InVitrogen Corporation), the AURI-C gene which confersresistance to the antibiotic aureobasidin A, phosphinothricin-resistancegene, neomycin phosphotransferase gen (nptII), hygromycin-resistancegene, ∃-glucuronidase (GUS) gene, chloramphenicol acetyltransferase(CAT) gene, green fluorescent protein-encoding gene or the luciferasegene, amongst others.

Preferably, the selectable marker gene is the nptII gene or Kan^(r) geneor green fluorescent protein (GFP)-encoding gene.

Those skilled in the art will be aware of other selectable marker genesuseful in the performance of the present invention and the subjectinvention is not limited by the nature of the selectable marker gene.

The present invention extends to all genetic constructs essentially asdescribed herein, which include further genetic sequences intended forthe maintenance and/or replication of said genetic construct inprokaryotes or eukaryotes and/or the integration of said geneticconstruct or a part thereof into the genome of a eukaryotic cell ororganism.

Standard methods described supra may be used to introduce the constructsinto the cell, tissue or organ, for example, liposome-mediatedtransfection or transformation, transformation of cells with attenuatedvirus particles or bacterial cells, cell mating, transformation ortransfection procedures known to those skilled in the art.

Additional means for introducing recombinant DNA into plant tissue orcells include, but are not limited to, transformation using CaCl₂ andvariations thereof, direct DNA uptake into protoplasts, PEG-mediateduptake to protoplasts, microparticle bombardment, electroporation,microinjection of DNA, microparticle bombardment of tissue explant orcells, vacuum-infiltration of tissue with nucleic acid, or in the caseof plants, T-DNA-mediate transfer from Agrobacterium to the planttissue.

For microparticle bombardment of cells, a microparticle is propelledinto a cell to produce a transformed cell. Any suitable ballistic celltransformation methodology and apparatus can be used in performing thepresent invention. Exemplary apparatus and procedures are disclosed byStomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat.No. 4,945,050). When using ballistic transformation procedures, thegenetic construct may incorporate a plasmid capable of replicating inthe cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to5 μm gold spheres. The DNA construct may be deposited on themicroparticle by any suitable technique, such as by precipitation.

In a further embodiment of the present invention, the genetic constructsdescribed herein are adapted for integration into the genome of a cellin which it is expressed. Those skilled in the art will be aware that,in order to achieve integration of a genetic sequence or geneticconstruct into the genome of a host cell, certain additional geneticsequences may be required. In the case of plants, left and right bordersequences from the T-DNA of the Agrobacterium tumefaciens Ti plasmidwill generally be required.

According to another aspect of the invention, there is provided atransformed plant cell containing an expression vector as broadly hereindescribed. The term “plant cell” as used herein refers to protoplasts orother cells derived from plants, gamete-producing cells, and cells whichregenerate into whole plants. Plant cells include cells in plants aswell as protoplasts or other cells in culture. By “plant tissue” ismeant differentiated and undifferentiated tissue derived from roots,shoots, pollen, seeds, tumour tissue, such as crown galls, and variousforms of aggregations of plant cells in culture, such as embryos andcalluses.

In a still further aspect, the invention provides a regenerateddifferentiated plant consisting of plant cells containing an expressionvector as broadly herein described. Plants may conveniently beregenerated from transformed plant cells or tissues or organs onhormone-containing media and the regenerated plants may take a varietyof forms, such as chimeras of transformed cells and non-transformedcells; clonal transformants (e.g. all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissue(e.g. a transformed root stock grafted to an untransformed scion incitrus species). Transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plants may be selfedto give homozygous second generation (or T2) transformed plants, and theT2 plants further propagated through classical breeding techniques.

Accordingly, yet another aspect of the invention is directed to aregenerated differentiated plant comprising a nucleic acid moleculeencoding a polypeptide comprising an amino acid sequence substantiallyas set forth in SEQ ID NO:2 or an amino acid sequence having at leastabout 71% similarity to SEQ ID NO:2, wherein said polypeptide is presentin plant zygotic embryos or embryogenic callus and is substantially notpresent in non-embryogenic tissue.

In a related embodiment, the present invention is directed to aregenerated differentiated plant comprising a nucleic acid molecule,wherein the nucleic acid molecule comprises a sequence of nucleotidessubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form, or a nucleotide sequence having at least about 71%similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form or anucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3or its complementary form under low stringency conditions.

As used herein, “plant” and “differentiated plant” refer to a wholeplant or plant part containing differentiated plant cell types, tissuesand/or organ systems. Plantlets and seeds are also included within themeaning of the foregoing terms. Plants included in the invention are anyplants amenable to transformation techniques, including angiosperms,gymnosperms, monocotyledons and dicotyledons. In a most preferredembodiment, the plant is oil-palm or a related plant.

In yet another aspect, the invention provides oil-palm harvested from adifferentiated plant as broadly described above.

The nucleic acid molecule of the present invention is useful, interalia, to distinguish embryogenic from non-embryogenic material.Accordingly, embryogenic material may be detected in vitro by screeningfor expression of the subject nucleic acid molecule. As defined above,expression may result in transcript or translation product or both. Arange of assays may be employed to detect nucleic acid transcript ortranslation products. These assays are well known to those skilled inthe art and particularly useful assays are described below.

Accordingly, one aspect of the present invention contemplates a methodfor detecting embryogenic plant material, said method comprisingscreening for expression of a nucleic acid molecule, said nucleic acidmolecule comprising a sequence of nucleotides substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or a nucleotidesequence having at least about 71% similarity to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions wherein expression of said nucleic acidmolecule is indicative of the presence of embryogenic material.

Reference to “material” includes reference to cells, tissues, callusand/or organelles or related tissue. The expression “detecting”embryogenic plant material includes distinguishing between embryogenicand non-embryogenic material.

The assay may be conducted in any number of ways. For example, mRNAtranscript may be detected as the expression product. In one method,Northern blot analysis may be used.

According to this embodiment, there is provided a method for detectingembryogenic plant material, said method comprising immobilizing a sampleputatively containing RNA from the material to be screened on a solidsupport and contacting said immobilized RNA with a labelled nucleotidesequence capable of hybridizing to all or part of an mRNA transcriptcorresponding to the nucleotide sequence set forth in SEQ ID NO:1 or SEQID NO:3 or their derivatives or homologues as defined herein and thendetecting the presence of said label.

The label may be any reporter molecule capable of providing anidentifiable signal such as ³²P, ³⁵S, or other radionucleotide,fluorogenic molecule, enzyme or other suitable reporter molecule.

By “immobilized” means both a “dot blot” type assay or anelectrophoretic assay where the total RNA is subjected toelectrophoresis.

The probe is preferably a cDNA molecule including a fragment (e.g. fromabout 8 nucleotides in length) or whole or substantially whole lengthmolecules corresponding to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form. Alternatively, the probe is a RNA moleculecomplementary to the target mRNA sequence.

Any number of variations may be performed to the assay without departingfrom the scope or spirit of the invention.

In another embodiment, expression is determined by detecting thetranslation product, i.e. a sequence of amino acids such as in the formof a peptide, polypeptide or protein (encompassed herein by the term“polypeptide”).

In one useful embodiment, antibodies are generated to the subjectpolypeptide. Such antibodies may be used in an immunoassay to detect theinstant polypeptide. The presence of the polypeptide is indicative ofembryogenic material.

Accordingly, another aspect of the present invention contemplates anantibody to a polypeptide, said polypeptide comprising a sequence ofamino acids substantially as set froth in SEQ ID NO:2 or an amino acidsequence having at least about 71% similarity to SEQ ID NO:2, whereinsaid polypeptide is present in plant zygotic embryos or embryogeniccallus and is substantially not present in non-embryogenic tissue.

Either monoclonal or polyclonal antibodies may be employed. The use ofmonoclonal antibodies in an immunoassay is particularly preferredbecause of the ability to produce them in large quantities and thehomogeneity of the product. The preparation of hybridoma cell lines formonoclonal antibody production derived by fusing an immortal cell lineand lymphocytes sensitized against the immunogenic preparation can bedone by techniques which are well known to those who are skilled in theart. (See, for example, Douillard and Hoffman, (1981); Kohler andMilstein, (1975); (1976).

Another aspect of the present invention contemplates a method fordetecting a polypeptide which is indicative of the presence ofembryogenic tissue in oil-palm or related plants, said method comprisingcontacting the tissue or an extract thereof with an antibody specificfor said polypeptide or its derivatives or homologues for a time andunder conditions sufficient for an antibody-polypeptide complex to form,and then detecting said complex.

The presence of the polypeptide may be detected in any number of wayssuch as by Western blotting and ELISA procedures. A wide range ofimmunoassay techniques are available as can be seen by reference to U.S.Pat. Nos. 4,016,043, 4,424,279 and 4,018,653.

In one assay, an unlabelled antibody specific to the oil-palmpolypeptide is immobilized on a solid substrate and the sample to betested brought into contact with the bound molecule. After a suitableperiod of incubation, for a period of time sufficient to allow formationof an antibody-polypeptide complex, a second antibody specific to thepolypeptide, labelled with a reporter molecule capable of producing adetectable signal, is then added and incubated, allowing time sufficientfor the formation of another complex of antibody-polypeptide-labelledantibody. Any unreacted material is washed away, and the presence of thepolypeptide is determined by observation of a signal produced by thereporter molecule. The results may either be qualitative, by simpleobservation of the visible signal, or may be quantitated by comparingwith a control ample containing known amounts of polypeptide. Variationson this assay include a simultaneous assay, in which both sample andlabelled antibody are added simultaneously to the bound antibody. Thesetechniques are well known to those skilled in the art, including anyminor variations as will be readily apparent. In accordance with thepresent invention, the sample is one which might contain the oil-palmpolypeptide including cell or callus extract or lysate. The sample is,therefore, generally a biological sample.

In this assay, a first antibody having specificity for the polypeptideor antigenic parts thereof, is either covalently or passively bound to asolid surface. The solid surface is typically glass or a polymer, themost commonly used polymers being cellulose, polyacrylamide, nylon,polystyrene, polyvinyl chloride or polypropylene. The solid supports maybe in the form of tubes, beads, discs of microplates, or any othersurface suitable for conducting an immunoassay. The binding processesare well-known in the art and generally consist of cross-linkingcovalently binding or physically adsorbing, the polymer-antibody complexis washed in preparation for the test sample. An aliquot of the sampleto be tested is then added to the solid phase complex and incubated fora period of time sufficient (e.g. 2-40 minutes or overnight if moreconvenient) and under suitable conditions (e.g. from room temperature toabout 38° C. such as 25° C.) to allow binding of the antibody. Followingthe incubation period, the antibody solid phase is washed and dried andincubated with a second antibody specific for a portion of thepolypeptide. The second antibody is linked to a reporter molecule whichis used to indicate the binding of the second antibody to thepolypeptide.

An alternative method involves immobilizing the target molecules in thebiological sample and then exposing the immobilized target to specificantibody which may or may not be labelled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labellingwith the antibody.

Alternatively, a second labelled antibody, specific to the firstantibody is exposed to the target-first antibody complex to form atarget-first antibody-second antibody tertiary complex. The complex isdetected by the signal emitted by the reporter molecule.

By “reporter molecule”, as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay (ETA), an enzyme is conjugated tothe second antibody, generally by means of glutaraldehyde or periodate.As will be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, -galactosidase and alkaline phosphatase, amongst others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody-polypeptide complex, allowed tobind, and then the excess reagent is washed away. A solution containingthe appropriate substrate is then added to the complex ofantibody-polypeptide-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of hapten which was present in the sample.“Reporter molecule” also extends to use of cell agglutination orinhibition of agglutination such as red blood cells on latex beads, andthe like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-polypeptide complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength, the fluorescence observed indicatesthe presence of the hapten of interest Immunofluorescene and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

The present invention, therefore, provides in one embodiment, ascreening procedure to identify, detect or otherwise discriminatebetween embryogenic and non-embryogenic material. In this regard,substantial savings in time and cost may be made by removingnon-embryogenic material from tissue being used in in vitromultiplication of oil-palm or related plants. The ability to distinguishembryogenic cultures from the non-embryogenic cultures at an early stagefacilitates culling of cultures and thus expensive laboratory space canbe saved as well as months of labor. Furthermore, the ability to controlembryogenesis would meant that cultures need not depend on random chanceto attain their embryogenic potential. The assay of the presentinvention may also be automated or semi-automated where one or moresteps are controlled by a computer programme. Alternatively or inaddition, the present invention further provides a test kit foridentifying embryogenic material, said test kit in compartmental formcomprises in one compartment, an agent for detecting a nucleic acid orpolypeptide associated with embryogenic material in oil-palm plants orrelated plants; a second or further compartments are adapted to containreagents including solid supports for detecting the subject nucleic acidmolecule or polypeptide.

The polypeptide of the present invention is also useful in therapeutictreatments, such as in anti-aging.

Accordingly, another aspect of the present invention contemplates acomposition such as a pharmaceutical composition comprising thepolypeptide having an amino acid sequence as set forth in SEQ ID NO:2 ora functional homologue thereof or a molecule having at least 71%similarity to SEQ ID NO:2 and one or more pharmaceutically acceptablecarriers and/or diluents.

The preferred composition of the present invention is in the form of apharmaceutical composition.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dilution mediumcomprising, for example, water, ethanol, polyol (for example, glycerol,propylene glycol and liquid polyethylene glycol, and the like), suitablemixtures thereof and vegetable oils. The proper fluidity can bemaintained, for example, by the use of superfactants. The preventions ofthe action of microorganisms can be brought about by variousanti-bacterial and anti-fungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with theactive ingredient and optionally other active ingredients as required,followed by filtered sterilization or other appropriate means ofsterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, suitable methods of preparation includevacuum drying and the freeze-drying technique which yield a powder ofactive ingredient plus any additionally desired ingredient.

When the active ingredient is suitably protected, it may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet or administered via breast milk. Fororal therapeutic administration, the active ingredient may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafersand the like. Such compositions and preparations should contain at least1% by weight of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 5 to about 80% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between about 0.1 μg and 200 mg of active compound.Alternative dosage amounts include from about 1 μg to about 1000 mg andfrom about 10 μg to about 500 mg. These dosages may be per individual orper kg body weight. Administration may be per hour, day, week, month oryear.

The tablets, troches, pills, capsules, creams and the like may alsocontain the components as listed hereafter. A binder such as gum,acacia, corn starch or gelatin; excipients such as dicalcium phosphate;a disintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, anti-bacterial and anti-fungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art and except insofar as any conventional media or agent isincompatible with the active ingredient, their use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is particularly advantageous to incorporate the active ingredient asa cream capable of preventing or delaying aging.

The present invention is further described by the following non-limitingExamples.

Example 1 Reverse Transcription of PCR (RT-PCR)

A one-step RT-PCR was carried out with the use of the Titan One TubeRT-PCR System from Boehringer Mannheim.

The reaction components for master mix 1 and master mix 2 were preparedin separate DEPC-treated tubes. The components in master mix 1 were: 4μl of dNTP mix (10 mM)+1 μl downstream primer (10 μM)+1 μl upstreamprimer (10 μM)+1 μl template RNA (˜1 μg zygotic embryo total RNA)+2.5 μlDTT solution (100 mM)+7 μl RNase Inhibitor (40 U/μl)+sterile H₂O to afinal volume of 25 μl. The primers used here were the AGL15AtF(5′-AGGAGGATTGTGCAGAG-3′ [SEQ ID NO:5]) and AGL15AtR(5′-CAAACTCTCAGCTAGGCA-3′ [SEQ ID NO:6]) based on Heck et al. (1995).

In master mix 2, 10 μl of the 5×RT-PCR buffer with Mg²⁺ and 1 μl of theenzyme mix (AMY reverse transcriptase+Expand High Fidelity enzyme mix)were added together in a total volume of 25 μl made up by H₂O. The whole25 μl of master mix 1 and master mix 2 were added into a 0.2 mlthin-walled PCR tube on ice. The contents were mixed properly andbriefly centrifuged to collect the sample at the bottom of the tube. Thepreparation was overlaid with 30 μl mineral oil and placed in athermocycler, which had been equilibrated at 50° C. for 30 min, afterwhich the programme went directly into thermocycling.

The parameters had been set at: 94° C. for 2 min; 10 cycles at (94° C.for 30 sec; 50° C. for 30 sec; 68° C. for 45 sec); 25 cycles at (94° C.for 30 sec; 50° C. for 30 sec; 68° C. for 45 sec+cycle elongation of 5sec for each cycle); a final extension at 68° C. for 7 min. The RT-PCRproduct was analyzed on a 2% agarose gel. Bands were excised from thegel and purified using the Concert Rapid Gel Extraction kit (Clontech)and cloned into pCRScript (Stratagene). This cloning kit is very similarto the Zero Blunt TOPO PCR cloning kit except that clones are selectedon LB+50 μg/ml ampicillin medium.

Two bands were obtained with AGL15AtF and AGL15AtR (FIG. 1). Both bandswere excised, the ends were polished and they were then cloned intopCR-Script (Stratagene) and transformed into DH5α competent cells. Thesuccessfully transformed clones were designated RTPCR1 (for clone fromfragment 1) and RTPCR2 (for clone from fragment 2). RTPCR1 and RTPCR2were cultured and plasmid minipreps were done.

Example 2 Sequence Analysis

Clones to be sequenced were sent to ACGT (USA) and the sequencing wasperformed on a single-run basis using universal primers T3/T7 for mostclones except for clones from the enriched library, where the primersPN1/PN2 were used. Sequence analyses were carried out using DNASIS(Hitachi software package, 1997) and BLAST (Basic Local Alignment SearchTool) (Altshul et al., 1990; 1997), available via the internet athttp://www.ncbi.nlm.nih.gov. To analyze the sequences with their closelyrelated counterparts, the alignment of the sequences was done using theCLUSTALW programme available from the Biology Workbench version 3.2 athttp://biology.nesa.uiuc.edu.

Both analyses gave similar results. RTPCR1 was shown to have about 71%homology (DNASIS) to an embryo specific mRNA of Bromus secalinas, whichis found to be up-regulated in hydrated dormant seeds (Goldmark et al.,1992), as well as a few other dormancy related genes. RTPCR2, on theother hand was found to have a homology (>71%) to an S-phase specificgene (Uchimiya et al., 1994) which is involved in the cell cycle.Through the sequencing results, it was realized that the reason two verydistinct bands were produced was because each band was generated by onlyone type of primer. That is, RTPCR1 by AGL15AtR and RTPCR2 by AGL15AtF.

Example 3 Northern Hybridization

The different types of poly A⁺ RNA extracted were electrophoresed on 2%v/v formaldehyde gels and transferred onto nylon membranes usingstandard blotting techniques (Maniatis et al., 1982). Two percentformaldehyde gels (120 ml) were prepared by melting down 3 g of agarosein 110 ml of DEPC-treated H₂O and 15 ml 10×MOPS buffer (200 mM MOPS (Ph7.0), 10 mM EDTA, 50 mM sodium acetate). When the mixture cooled down toabout 55° C., 37% formaldehyde was added to a final concentration of 6%.The contents were properly mixed and then poured into the gel castingtray and allowed to set.

In the preparation of the RNA samples for analysis, about 10 to 15 μg oftotal RNA was used for each different type of tissue. The RNA samplesused were in a small volume of H₂O. At times, the samples needed to beconcentrated: the maximum volume of RNA that can be accommodated was 4.8μl for each preparation. In each tube, the required amount of RNA (ifless than 4.8 μl was needed, H₂O was added to a final volume of 4.8 μl)was added into 2 μl of 10×MOPS buffer, 3.2 μl of 37% formaldehyde and 10μl of formamide, making the total volume 20 μl. The samples were placedin a heating block at 65° C. for 15 min and immediately chilled on ice.Just before loading, 2 μl of loading buffer (50% glycerol+1 mMEDTA+0.25% bromophenol blue+0.25% xylene cyanole) was added into eachtube. Gels were electrophoresed in 1×MOPS buffer at a low voltage (20 to30V) until the bromophenol blue dye reached the bottom of the gel.

The gel was stained with 0.5 μg/ml ethidium bromide in 200 mM ammoniumacetate for 45 min to 1 hr. This was followed by destaining with severalchanges of DEPC-treated H₂O, until the bands of the RNA marker (GibcoBRL) and the rRNA of the samples were clearly visible. The individualbands of the marker were marked by making a hole in them. Similarly, therRNA bands (28S and 18S) of the samples were also randomly marked. Thegels were rinsed with DEPC-treated H₂O several times to remove theformaldehyde, followed by a final rinse with 2×SSC before blotting. Thegel was placed on its reverse side on the blotting apparatus, makingsure that no bubbles were trapped in between the gel and the wick, whichwas made of 2 pieces of 3MM Whatman chromatography paper that had beencut to size. The wick forms a bridge on a glass plate placed across acontainer with 10×SSC as the transfer buffer. A positively charged nylonmembrane was cut to size, along with 4 pieces of 3MM Whatmanchromatography paper of the same size, and pre-wetted in 2×SSC. First,the wet membrane was placed carefully onto the gel, without trapping anyair bubbles, and this was followed by the pre-wetted paper. Another 4pieces of dry 3MM Whatman chromatography paper and a stack of papertowels were placed over this. A glass plate was placed right at the topof this set-up and weights were added to keep them in direct contactwith each other. The transfer was allowed to occur for at least 16 hr.

After the transfer was completed, the blotting set-up was dismantled andthe marks previously made on the gel were penciled onto the membrane.The membrane was then rinsed in 2×SSC for 15 min and auto-crosslinked at120,000 μl of UV energy or alternatively baked at 80° C. for 1.5 to 2hr.

The probes were radioactively labeled using the High Prime kit(Boehringer Mannheim). As a control, 18S rRNA probe was also hybridizedto the RNA blots. The 18S rRNA probe was prepared by double-digestingthe pBG35 plasmid (Malaysian Palm Oil Board) with KpnI (Promega) andEcoR1 (Promega) at 37° C. overnight. The digest contained 1.0 μl plasmidpBG35, 2.0 μl 10× restriction buffer, 1.5 μl EcoR1 (12 U/μl), 1.5 μlKpn1 (12 U/μl) and sterile H₂O to a final volume of 20 μl. The digestionwas electrophoresed on a 0.8% agarose gel and the desired 1.6 kb bandwas excised from the gel and purified. Usually in the case of Northernanalysis, high stringency washes were applied (up to 0.5×SSC+0.1% SDS at65° C. for 15 min).

An initial, simple Northern analysis was carried out on the two RTPCRclones and interestingly, RTPCR1 was found to be expressed only inzygotic embryo (ZE) and embryogenic callus (EC), indicating that it maybe an embryogenic related gene. By comparison, RTPCR2 was constitutivelyexpressed (FIG. 1 b).

Example 4 (a) Further Characterization: Sequence of Full-Length Clone

Because of the interesting results obtained with RTPCR1, this clone wasfurther studied. RTPCR1 was only of partial length when obtained throughRT-PCR, therefore the zygotic embryo cDNA library was screened to obtainits full-length clone. The resultant clone was named as OPEm1. Thenucleotide and deduced amino acid sequences of OPEm1 is shown in FIG. 3.It contains an open reading frame (ORF) from position 30 to 605 encodinga protein with 192 amino acids. A hydrophobic region occurs close to thecarboxyl-terminus of the predicted protein, which has a predicted pI of7.48 (FIG. 2 b).

(b) Detailed Northern Analysis

A more detailed Northern blot was prepared to reconfirm the previousresults obtained (FIG. 2 a). The transcript size of OPEm1 was determinedto be approximately 1995 nucleotides (nt). The expression pattern ofOPEm1 further proves that this clone has the potential to be exploitedas an embryogenic marker because its expression can be detected in allthe embryogenic calli regardless of their clonal differences, insuspension cultures (lanes 9 and 10), in embryoids (lanes 11 and 12)right up to somatic embryos in the form of bipolar structures (lane 13).Expression signals in lanes 9, 10 and 13 are a little faint but werestill able to be visibly detected on the autoradiograph. Expression wasalso detected in zygotic embryo (lane 15). The expression of OPEm1 wasnot found in cultures that had lost their embryogenic potential (lanes2, 4 and 7), non-embryogenic calli (lanes 5 and 8) as well as othervegetative tissues such as the meristem, inflorescences and youngunexpended leaves of the oil-palm (lanes 16, 17 and 18 respectively).

Example 5 Southern Hybridization

Genomic DNA of the oil-palm extracted from young unexpanded leave. Thegenomic DNA (5 to 10 μg) was digested with several different enzymes.Each digestion contained 30 μl of genomic DNA (10 μg), 5 μl of 10×restriction buffer, 5 μl of BSA (1 mg/ml), 5 μl enzyme (EcoR1, BamH1,Hind III, Kpn1, Not1, Sfi1, Spe1 and Stu1) and sterile H₂O to a finalvolume of 50 μl. The digestion was carried out at 37° C. and completedigestion was ensured by an overnight incubation. The digested DNA waselectrophoresed on a 1.0% agarose gel along side a 1 kb DNA molecularweight marker (Promega). After the run, the gel was photographed andholes were made to mark the positions of the bands belonging to themarker. The gel was then immersed in depurination solution (0.25 N HCl)for 10 min with gentle shaking. The solution was decanted and the gelwas rinsed several times with sterile H₂O, after which denaturationsolution (0.5 M NaOH, 1.5 M NaCl) was added to the gel and agitated for30 min. This was again followed by several rinses of sterile H₂O andfinally the gel was neutralized with the neutralization solution (3 MNaCl, 0.5 M Tris-HCl (Ph 7.4)) for 30 min.

A similar blotting apparatus as for the Northern was set up for Southernanalysis. Southern analysis showed that OPEm1 gene may be a member of amultigene family (FIG. 2 c).

Example 6 3D-Structure

Since the results of the Northern analyses seems to contradict thefunction inferred for the gene based on its sequence similarity todormancy-related genes, it was decided that the protein structure ofOPEm1 should be determined, in order to try to elucidate the possiblefunction of the gene. FIG. 4 a represents the 3-dimensional (3D)structure predicted for OPEm1 and it was evident that it had a verysimilar structure to the monomer unit of a human peroxiredoxin (Choi etal., 1998). The human peroxiredoxin (Prx), C19S-hORF6, exists in theform of a homodimer (FIG. 4 c).

The structure of OPEm1 can be explained based on the 3D structure andtopology diagram of the C19S-hORF6 which is shown in FIGS. 4 b and 4 d,respectively. The monomer can be divided into two domains, D1 and D2.D1, the larger of the two, is the N-terminal, which contains thethioredoxin fold (active site of the enzyme). This site has a βαβ motifcomprised of four-stranded β-sheets (β3, β4, β6 and β7) and threeflanking α-helices (α2, α4 and α5). There are also two β-strands (β1 andβ2) and a short α-helix (α1) at the N-terminus just before thethioredoxin fold. After the βαβ motif, an α-helix (α3) and a β-strand(β5) are inserted. In the case of the C19S-hORF6, D2 comprises threeβ-strands and one α-helix and it is connected to D1 by the extendedhelix α5 and a following loop. In OPEm1, this region is very short,having only two β-sheets (8β and 9β) with a loop between them. Thisdifference is also reflected in FIG. 6, in which, by residue 189, theiramino acid sequences no longer show similarity with the group.

Example 7 Sequence Alignments

The deduced amino acid sequence of OPEm1 was compared with other plantPrx sequences that have been isolated. FIG. 5 shows the alignment ofamino acid sequences 1-Cys and 2-Cys groups of Prx in plants. It seemsthat OPEm1 is more closely related to 1-Cys rather than to the 2-Cysgroup of Prx. This is reflected in FIG. 6, which shows an alignmentbetween OPEm1 and other 1-Cys Prx in plants. They share similarsequences surrounding the first cysteine, which also sets the 1-Cysapart from the 2-Cys group. In 1-Cys, the sequences are PVCT, whereas in2-Cys, they are represented by FVCP. From FIG. 6, it was also observedthat OPEm1's C-terminus differs from the other 1-Cys Prx group. Based onthe hydropathy plot (FIG. 2 b), OPEm1 may be membrane bound at thisregion, unlike the other 1-Cys members. However, the same region in1-Cys Prx members indicates the presence of a nuclear localizationsignal that facilitates the nuclear targeting function of the proteinthat is missing from OPEm1.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

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1. An isolated nucleic acid molecule encoding a polypeptide comprisingan amino acid sequence substantially as set forth in SEQ ID NO:2 or anamino acid sequence having at least about 71% similarity to SEQ ID NO:2,wherein said polypeptide is present in plant zygotic embryos orembryogenic callus and is substantially not present in non-embryogenictissue.
 2. An isolated nucleic acid molecule of claim 1, wherein saidnucleic acid molecule comprises a sequence of nucleotides substantiallyas set forth in SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, ora nucleotide sequence having at least about 71% similarity to SEQ IDNO:1 or SEQ ID NO:3 or its complementary form or a nucleotide sequencecapable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form under low stringency conditions.
 3. An isolatednucleic acid molecule of claim 1, wherein the nucleic acid molecule isdevelopmentally regulated.
 4. An isolated nucleic acid molecule of claim1, 2 or 3, wherein the nucleic acid molecule is expressed substantiallyin embryogenic material of oil-palm plants or related plants but not innon-embryogenic material.
 5. An isolated nucleic acid molecule of claim1, wherein the nucleic acid molecule comprises the nucleotide sequencesubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3.
 6. An isolatednucleic acid molecule of claim 1, wherein the nucleic acid moleculecomprises the nucleotide sequence substantially as set forth in SEQ IDNO:1.
 7. An isolated nucleic acid molecule of claim 1, wherein thenucleic acid molecule comprises the nucleotide sequence substantially asset forth in SEQ ID NO:3.
 8. A genetic construct comprising a nucleicacid molecule encoding a polypeptide comprising an amino acid sequencesubstantially as set forth in SEQ ID NO:2 or an amino acid sequencehaving at least about 71% similarity to SEQ ID NO:2, wherein saidpolypeptide is present in plant zygotic embryos or embryogenic callusand is substantially not present in non-embryogenic tissue.
 9. A geneticconstruct of claim 8, wherein the nucleic acid molecule is substantiallyas set forth in SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, ora nucleotide sequence having at least about 71% similarity to SEQ IDNO:1 or SEQ ID NO:3 or its complementary form or a nucleotide sequencecapable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form under low stringency conditions.
 10. A geneticconstruct of claim 8, wherein the nucleic acid molecule isdevelopmentally regulated.
 11. A genetic construct of claim 8, 9 or 10,wherein the nucleic acid molecule is expressed substantially inembryogenic material of oil-palm plants or related plants but not innon-embryogenic material.
 12. A genetic construct of claim 8, whereinthe nucleic acid molecule comprises a sequence of nucleotidessubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3.
 13. A geneticconstruct of claim 12, wherein the nucleic acid molecule comprises asequence of nucleotides substantially as set forth in SEQ ID NO:1.
 14. Agenetic construct of claim 12, wherein the nucleic acid moleculecomprises a sequence of nucleotides substantially as set forth in SEQ IDNO:3.
 15. A genetic construct of claim 8 or 9, wherein said constructfurther comprises one or more promoter sequences or transcriptiontermination sequences.
 16. A genetic construct of claim 15, wherein saidconstruct further comprises one or more origins of replication and/orselectable marker gene sequences.
 17. A vector comprising a construct ofany one of claims 8 to
 16. 18. A host cell comprising a nucleic acidmolecule encoding a polypeptide comprising an amino acid sequencesubstantially as set forth in SEQ ID NO:2 or an amino acid sequencehaving at least about 71% similarity to SEQ ID NO:2, wherein saidpolypeptide is present in plant zygotic embryos or embryogenic callusand is substantially not present in non-embryogenic tissue.
 19. A hostcell of claim 18, wherein said nucleic acid molecule comprises asequence of nucleotides substantially as set forth in SEQ ID NO:1 or SEQID NO:3 or its complementary form, or a nucleotide sequence having atleast about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form or a nucleotide sequence capable of hybridizing toSEQ ID NO:1 or SEQ ID NO:3 or its complementary form under lowstringency conditions.
 20. A host cell of claim 18, wherein the nucleicacid molecule is developmentally regulated.
 21. A host cell of claim 18,19 or 20 wherein the nucleic acid molecule is expressed substantially inembryogenic material of oil-palm plants or related plants but not innon-embryogenic material.
 22. A host cell of claim 18, wherein thenucleic acid molecule comprises the nucleotide sequence substantially asset forth in SEQ ID NO:1 or SEQ ID NO:3.
 23. A host cell of claim 18,wherein the nucleic acid molecule comprises the nucleotide sequencesubstantially as set forth in SEQ ID NO:1.
 24. A host cell of claim 18,wherein the nucleic acid molecule comprises the nucleotide sequencesubstantially as set forth in SEQ ID NO:3.
 25. A host cell of claim 18,wherein the cell is a plant cell.
 26. A plant cell of claim 25, whereinthe cell is from an oil-palm plant.
 27. An isolated polypeptide orbiologically-active fragment thereof or a variant or derivative ofthese, said polypeptide comprising the amino acid sequence set forth inSEQ ID NO:2 or an amino acid sequence having at least about 71%similarity to SEQ ID NO:2, wherein said polypeptide is present in plantzygotic embryos or embryogenic callus and is substantially not presentin non-embryogenic tissue.
 28. An isolated polypeptide of claim 27,wherein the amino acid sequence is encoded by the nucleotide sequencesubstantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or a nucleotidesequence having at least about 71% similarity to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions.
 29. An isolated polypeptide of claim27, wherein the amino acid sequence is expressed substantially inembryogenic material of oil-palm plants or related plants but not innon-embryogenic material.
 30. An isolated polypeptide of claim 27 or 28,wherein the nucleotide sequence is substantially as set forth in SEQ IDNO:1.
 31. An isolated polypeptide of claim 27 or 28, wherein thenucleotide sequence is substantially as set forth in SEQ ID NO:3.
 32. Anisolated polypeptide of any one of claims 27 to 31, wherein thepolypeptide has antioxidant properties.
 33. An isolated polypeptide ofclaim 32, wherein the polypeptide is peroxiredoxin.
 34. A method forproducing a recombinant polypeptide in a host cell or tissue, saidmethod comprising introducing into the said cell or tissue an expressionvector comprising a nucleic acid molecule wherein said nucleic acidmolecule comprises a sequence of nucleotides substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or a nucleotidesequence having at least about 71% similarity to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form or a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formunder low stringency conditions, wherein said nucleic acid molecule isoperably linked to one or more regulatory sequences such that thenucleic acid molecule is capable of being expressed in said cell ortissue.
 35. A method of claim 34, wherein said expression vectorcomprises a genetic construct comprising a nucleic acid moleculecomprising a sequence of nucleotides encoding an amino acid sequencesubstantially as set forth in SEQ ID NO:2 or an amino acid sequencehaving at least about 71% similarity to SEQ ID NO:2.
 36. A method ofclaim 34, wherein said nucleic acid molecule comprises a sequence ofnucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3.37. A method of claim 36, wherein said nucleic acid molecule comprises asequence of nucleotides substantially as set forth in SEQ ID NO:1.
 38. Amethod of claim 36, wherein said nucleic acid molecule comprises asequence of nucleotides substantially as set forth in SEQ ID NO:3.
 39. Amethod for modulating apoptotic processes in a cell or tissue, saidmethod comprising introducing into said cell or tissue an expressionvector comprising a nucleic acid molecule, said nucleic acid moleculecomprising a sequence of nucleotides substantially as set forth in SEQID NO:1 or SEQ ID NO:3 or its complementary form, a nucleotide sequencehaving at least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 orits complementary form or a nucleotide sequence capable of hybridizingSEQ ID NO:1 or SEQ ID NO:3 or its complementary form under lowstringency conditions wherein said nucleic acid molecule is operablylinked to one or more regulatory sequences such that the nucleic acidmolecule is capable of being expressed in said cell or tissue.
 40. Amethod of claim 39, wherein said expression vector comprises a geneticconstruct comprising a nucleic acid molecule comprising a sequence ofnucleotides encoding an amino acid sequence substantially as set forthin SEQ ID NO:2 or an amino acid sequence having at least about 71%similarity to SEQ ID NO:2.
 41. A method of claim 39, wherein saidnucleic acid molecule comprises a sequence of nucleotides substantiallyas set forth in SEQ ID NO:1 or SEQ ID NO:3.
 42. A method of claim 41,wherein said nucleic acid molecule comprises a sequence of nucleotidessubstantially as set forth in SEQ ID NO:1.
 43. A method of claim 41,wherein said nucleic acid molecule comprises a sequence of nucleotidessubstantially as set forth in SEQ ID NO:3.
 44. A method for modulatingapoptotic processes in a cell, said method comprising administering tosaid cell an apoptotic process-controlling effective amount of arecombinant polypeptide, said polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2 or an amino acid sequence having atleast about 71% similarity to SEQ ID NO:2, said administration being fora time and under conditions sufficient to modulate apoptosis.
 45. Amethod for detecting embryogenic plant material, said method comprisingimmobilizing a sample putatively containing RNA from the material to bescreened on a solid support and contacting said immobilized RNA with alabelled nucleotide sequence capable of hybridizing to all or part of anmRNA transcript corresponding to the nucleotide sequence set forth inSEQ ID NO:1 or SEQ ID NO:3 or their derivatives or homologues as definedherein and then detecting the presence of said label.
 46. A method ofclaim 45, wherein said nucleotide sequence is substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3.
 47. A method of claim 45, wherein saidnucleotide sequence is substantially as set forth in SEQ ID NO:1.
 48. Amethod of claim 45, wherein said nucleotide sequence is substantially asset forth in SEQ ID NO:3.
 49. An antibody to a polypeptide, saidpolypeptide comprising a sequence of amino acids substantially as setforth in SEQ ID NO:2 or an amino acid sequence having at least 71%similarity to SEQ ID NO:2, wherein said polypeptide is present in plantzygotic embryos or embryogenic callus and is substantially not presentin non-embryogenic tissue.
 50. An antibody of claim 49, wherein theamino acid sequence is encoded by a nucleotide sequence substantially asset forth in SEQ ID NO:1 or SEQ ID NO:3 or a nucleotide sequence havingat least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form or a nucleotide sequence capable of hybridizing toSEQ ID NO:1 or SEQ ID NO:3 or its complementary form under lowstringency conditions.
 51. An antibody of claim 49, wherein the aminoacid sequence is encoded by a nucleotide sequence substantially as setforth in SEQ ID NO:1 or SEQ ID NO:3.
 52. An antibody of claim 49,wherein the nucleotide sequence is substantially as set forth in SEQ IDNO:1.
 53. An antibody of claim 49, wherein the nucleotide sequence issubstantially as set forth in SEQ ID NO:3.
 54. An antibody of claim 49,wherein the polypeptide has antioxidant properties.
 55. An antibody ofclaim 54, wherein the polypeptide is peroxiredoxin.
 56. A method fordetecting a polypeptide which is indicative of the presence ofembryogenic tissue in oil-palm or related plants, said method comprisingcontacting the tissue or an extract thereof with an antibody specificfor said polypeptide or its derivatives or homologues for a time andunder conditions sufficient for an antibody-polypeptide complex to form,and then detecting said complex.
 57. A method of claim 56, wherein thepolypeptide comprises a sequence of amino acids substantially as setforth in SEQ ID NO:2 or an amino acid sequence having at least 71%similarity to SEQ ID NO:2.
 58. A method of claim 57, wherein the aminoacid sequence is encoded by a nucleotide sequence substantially as setforth in SEQ ID NO:1 or SEQ ID NO:3 or a nucleotide sequence having atleast about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or itscomplementary form or a nucleotide sequence capable of hybridizing toSEQ ID NO:1 or SEQ ID NO:3 or its complementary form under lowstringency conditions.
 59. A method of claim 56, wherein the amino acidsequence is encoded by a nucleotide sequence substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3.
 60. A method of claim 56, wherein theamino acid sequence is encoded by a nucleotide sequence substantially asset forth in SEQ ID NO:1.
 61. A method of claim 56, wherein the aminoacid sequence is encoded by a nucleotide sequence substantially as setforth in SEQ ID NO:3.
 62. A pharmaceutical composition comprising thepolypeptide having an amino acid sequence as set forth in SEQ ID NO:2 ora functional homologue thereof or a molecule having at least 71%similarity to SEQ ID NO:2 and one or more pharmaceutically-acceptablecarriers and/or diluents.
 63. A pharmaceutical composition of claim 62,wherein the amino acid sequence is as set forth in SEQ ID NO:2.
 64. Aregenerated differentiated plant comprising a nucleic acid moleculeencoding a polypeptide comprising an amino acid sequence substantiallyas set forth in SEQ ID NO:2 or an amino acid sequence having at leastabout 71% similarity to SEQ ID NO:2, wherein said polypeptide is presentin plant zygotic embryos or embryogenic callus and is substantially notpresent in non-embryogenic tissue.
 65. A regenerated differentiatedplant of claim 64, wherein nucleic acid molecule comprises a sequence ofnucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 orits complementary form, or a nucleotide sequence having at least about71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary formor a nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ IDNO:3 or its complementary form under low stringency conditions.
 66. Aregenerated differentiated plant of claim 64, wherein the amino acidsequence is encoded by a nucleotide sequence substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3.
 67. A regenerated differentiated plant ofclaim 64, wherein the amino acid sequence is encoded by a nucleotidesequence substantially as set forth in SEQ ID NO:1.
 68. A regenerateddifferentiated plant of claim 64, wherein the amino acid sequence isencoded by a nucleotide sequence substantially as set forth in SEQ IDNO:3.
 69. A regenerated differentiated plant of claim 64, wherein theplant is an oil palm plant.